Detection of Mycoplasma Pneumoniae by Polymerase Chain Reaction in Middle Ear Fluids From Infants With Acute Otitis Media

Acute otitis media (AOM) is often preceded by symptoms of upper respiratory tract infection (URI).1, 2 Although AOM itself is usually caused by bacteria,1, 3 URI is mostly of viral origin4 and viruses are commonly regarded as predisposing to the bacterial infection.5 The bacterial species most often detected in middle ear fluid (MEF) are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.1 Several other pathogenic bacteria have also occasionally been detected in MEF.3Mycoplasma pneumoniae, although a common respiratory pathogen, has been isolated only twice from MEF,6 plus once from the ear canal after tympanocentesis7 and once from a myringitis vesicle.8 This may partly be caused by difficulties in culturing this fastidious organism. Nevertheless otitis media is often mentioned as a clinical condition associated with serologically verified M. pneumoniae infection.9, 10 With the development of gene techniques better tools are now available for studying the role of M. pneumoniae in acute otitis media. Patients and methods. The patients belonged to the Finnish Otitis Media Cohort Study conducted from 1994 to 1997 (principal investigators, AK Takala and T Kilpi) in which a cohort of 329 children was followed up from 2 months to 2 years of age to investigate the etiology and epidemiology of URI among other risk factors for AOM. Whenever acute respiratory infection was suspected, the child had free access to a special study clinic. When AOM was diagnosed and confirmed by myringotomy, MEF was drawn from the inflamed ear(s). Nasopharyngeal aspirates (NPA) were also collected, as well as paired sera from most of the cases. The MEF and NPA samples were divided, frozen and distributed as frozen among a few laboratories. We had access to one-third of the MEF specimens of the Finnish Otitis Media Cohort Study, 380 unselected middle ear fluid specimens from 138 children. There were 77 MEF paired samples from bilaterally infected children and 226 single samples. NPA samples taken at the same visit as the MEF samples were available in 268 cases. Two hundred twenty-five serum pairs (fulfilling the predefined time criteria) were obtained for serologic analysis. DNA extraction. MEF and NPA specimens were thawed to room temperature; all of the material in the tubes was removed and the DNA was extracted with a commercial kit for DNA purification (QiaGen, Blood Kit). The method requires 200 μl of sample. Effusions with smaller volumes remaining were supplemented with PBS to a final volume of 200 μl before treatment. About 40 samples were treated during one working day, and the DNA amplification took place immediately afterward. DNA amplification. PCR for the detection of M. pneumoniae was performed with primers, which were selected from the P1 attachment protein of this bacterium.11 The primers were 5′-CAC CCG AAT GCT TTG GCC TTT CAG-3′ and 5′-CCC ATC TAA CAG TTC AGC GAG TGG-3′. With these primers the length of the PCR product was 569 bp. The PCR was performed in a total volume of 50 μl, and the volume of the DNA sample was 25 μl. The PCR mixture consisted of deoxyadenosine 5′-triphosphate, deoxycytidine 5′-triphosphate, deoxyguanosine 5′-triphosphate and thymidine 5′-triphosphate (0.2 mM each); 50 mM Tris-HCl (pH 8.8); 15 mM (NH4)2SO4; 1.5 mM MgCl2; 0.1% Triton X-100; 01% gelatin; 0.5 μM concentrations of each of the two primers and 2.5 units of Taq polymerase. After predenaturation at 95°C for 5 min, 35 amplification cycles were run. Each cycle consisted of a denaturation (30 s at 94°C), an annealing (1 min at 60°C) and an extension (3 min at 72°C) step. By taking a sample of 10 μl from each tube (one-fifth of the total volume), the PCR products were analyzed on 1.8% agarose gels containing 0.15 μg/ml of ethidium bromide. All the gels were blotted onto nylon filters (HyBond N; Amersham) and hybridized with a radioactive probe. The probe was prepared by PCR with primers that amplified a piece of 514 bp located internal to the first pair of primers. We have used the same oligonucleotides as nested primers in other connections (not published). The PCR product was purified and labeled with deoxycytidine 5′-[32P]triphosphate (Amersham) using a random-primed labeling kit (Boehringer Mannheim). As a positive control we used M. pneumoniae culture (strain ATCC 29342). Distilled water served as a negative control. After the primary results, the positive MEF samples were taken from the -70°C freezer, and the DNA amplification with hybridization and autoradiography was repeated. Serology. The complement fixation test was performed according to the principles of a standard micromethod.12 IgG and IgM specific antibodies against M. pneumoniae were determined by enzyme immunoassay.13 Results. Three hundred eighty middle ear fluids from infants with AOM were analyzed for M. pneumoniae with PCR and Southern blot hybridization. Altogether there were 16 hybridization positive results (4.2%) distributed among several autoradiographs. As seen in Fig. 1, which is a collection of 13 of the 16 positive samples, some specimens yielded several bands that were visible under UV light in the agarose gel, although in the initial assays a single band of the expected size was visible. However, the correct sizes of the radioactive bands seen after hybridization verified the M. pneumoniae positivity of the 16 samples. Seven of the 16 positive samples represented one of paired samples from both ears, the MEF from one ear being positive but the other being negative. No MEF pairs were both positive. Among the 16 patients with M. pneumoniae-positive results from MEF, the corresponding NPA samples were available in 7 cases; only 1 was positive with M. pneumoniae PCR. Among the 225 valid paired sera 4 significant titer rises were detected by complement fixation test, but in these cases the MEF samples were PCR-negative. Among the 16 PCR-positive cases there was no antibody titer increase but 4 titer decreases: 1 by complement fixation test; 3 by enzyme immunoassay. Other bacteria were found in 11 specimens:S. pneumoniae was found in 1, H. influenzae in 2 and Moraxella catarrhalis in 8 samples. Viruses were found in 3 samples: parainfluenza 3 virus in 1 and rhinoviruses in 2. The ages of the of the M. pneumoniae-positive patients ranged from 2 to 22 months (mean, 9.4; sd 5.5; median, 8.3.Fig. 1: Detection of M. pneumoniae from MEF samples by PCR. The PCR products were separated on a 1.8% agarose gel stained with ethidium bromide and photographed under UV light. A, collection of positive samples. Lanes M, 100-bp DNA ladder marker (Boehringer Mannheim); Lanes 1 to 13, MEF samples treated as described in “Patients and methods”; Lane 14, positive control. B, Southern blot of the same gel as in A followed by hybridization with a radioactive probe. The original patient samples contained different amounts of template. For autoradiography they had to be arranged in two groups; the first five had a shorter exposure time than the last eight lanes of the blots.Discussion. To our knowledge M. pneumoniae has been found by MEF culture only twice6 before the present study and never by PCR. Culture methods for M. pneumoniae are insensitive, probably resulting in underestimation of the prevalence of M. pneumoniae in MEF during AOM. We had the opportunity to examine MEF samples from children belonging to the well-characterized Finnish Otitis Media Cohort Study. According to the protocol, in the case of AOM the primary choice for antimicrobial agent was penicillin V or oral amoxicillin. For penicillin-sensitive children the choice was trimethoprim-sulfamethoxazole or cefaclor. None of these is effective against M. pneumoniae. However, the laborious isolation of M. pneumoniae was not attempted (scanty amounts of MEF available). With PCR M. pneumoniae was detected in 16 of 380 MEF specimens. The significance of this finding is not clear, but several speculations can be made. For example M. pneumoniae might be passively transported to the middle ear from the nasopharynx during inflammation. In a more active role M. pneumoniae could precede the bacterial AOM causing upper respiratory infection, like viruses. The tendency for antibody decreases seems to point in this direction. Furthermore M. pneumoniae could act as a cofactor with some other bacteria, enhancing their pathogenicity in causing AOM. Finally there is the possibility that M. pneumoniae itself causes AOM. If M. pneumoniae has an active role in the development of AOM or in prolonging recovery from the illness, this might affect the choice of optimal antimicrobial treatment. We shall continue our efforts to gather more information on the significance of M. pneumoniae in acute otitis media. Acknowledgments. We thank Annamari Harberg for performing the PCR reactions. We are grateful to Professor Maija Leinonen, who provided us with the samples after bacteriologic analysis, and to Professor Tapani Hovi, for his valuable comments.